Thioredoxin (TRX) is a 12 kDa redox cycling enzyme that is ubiquitously expressed in all cell types and is part of a key system for maintaining a reducing intracellular environment, working in a coordinate manner with thioredoxin reductase and NADPH (collectively referred to as the TRX system). TRX plays a role in regulating many different signaling processes, including cell cycle signaling, apoptosis, and glucose metabolism (Holmgren et al. (2010) Biochem Biophys Res Commun. 396, 120-124). Oxidative stress leads to TRX up-regulation, which then can be secreted by a yet-undefined leaderless pathway. TRX 80, a 10 KDa cleavage product of secreted TRX thought to be composed of the 80 N-terminal amino acids, is produced mainly from cleavage of secreted TRX by activated monocytes/macrophages (Silberstein et al. (1987) J Immunol 138, 3042-3050; Silberstein et al. (1989) J Immunol 143, 979-983). TRX 80 has monocyte chemoattractant activity and induces differentiation and activation of monocytes to a highly inflammatory phenotype that produces TNFα, IL-1β, IL-6 and IL-8, termed the TRX 80 activated monocyte (TAM) (Pekkari et al. (2005) Blood 105, 1598-1605). These cells also upregulate the costimulatory molecule CD86, and when cocultured with T cells produce IL-12 and aid in stimulating interferon-γ production (Pekkari et al. (2005) (supra); Pekkari et al. (2001) Blood 97, 3184-3190), suggesting that TAMs facilitate the generation of TH1 (proinflammatory) lymphocytes.
Increased plasma TRX levels have been demonstrated in many disease conditions, including heart failure, cardiomyopathy, cancer, asthma, and rheumatoid arthritis, among others, and are thought to be highly indicative of oxidative stress (Yoshida et al. (1999) J Immunol 163, 351-358; Kishimoto et al. (2001) Jpn Circ J 65, 491-494; Jikimoto et al. (2002) Mol Immunol 38, 765-772; Yamada et al. (2003) Immunol Lett 86, 199-205; Miwa et al. (2005) Circ J 69, 291-294; Grogan et al. (2003) Hum Pathol 31, 475-481). TRX has been tested as an oxidative stress marker in a study examining of the administration of A-type natriuretic peptide to reduce oxidative stress in heart failure patients (Shono et al. (2007) Circ J 71, 1040-1046).
Examination of TRX 80 levels in human disease or in model systems has been somewhat limited to date. The cleavage of TRX to TRX 80 may have profound influence on local sites of oxidative stress. TRX 80 levels were shown to be highly variable in plasma of a small number of presumed-healthy donors (Pekkari et al. (2000) J Biol Chem 275, 37474-37480). Additionally, production of TRX 80 by synoviocytes cultures from patients with rheumatoid arthritis (RA) has been demonstrated (Lemarechal et al. (2007) Clin Sci (Lond) 113, 149-155). Elevated levels of secreted TRX previously have been associated with RA (Jikimoto et al. (2002) (supra)), and in the aforementioned study by Lemarechal and colleagues, TRX 80 was produced at basal levels by cultured synovial cells obtained from RA patients, but not from osteoarthritis patients, with production increased by IL-1β and/or TNF-α stimulation. This suggests a role for TRX 80 production in the infiltration and proliferation of immune cells that are hallmarks of RA.
Several experimental approaches have been used to identify TRX and/or TRX 80 in biological samples, each with its own strengths and limitations. Due to their different functions, it is important to distinguish TRX from TRX 80. However, as TRX 80 is derived from the same amino acid sequence as TRX and they are relatively close in size (12 vs. 10 kDa), it can be difficult to clearly resolve them in a standard immunoblotting assay. Even antibodies developed to specifically recognize TRX 80 in its native nondenatured form may recognize both TRX 80 and TRX in western blot (Sahaf et al. (1997) Exp Cell Res 236, 181-192).
ELISA is frequently used to detect TRX. While potentially informative, most ELISAs may crossreact to TRX 80. One group has designed an ELISA that specifically detects the truncated form TRX 80. However, this assay still shows a very small level of cross reactivity for the full length protein (Pekkari et al. (2000) (supra)), which can be problematic if TRX 80 needs to be accurately monitored and is present at much lower levels than TRX.
Other TRX properties may contribute to inaccurate quantitation by immunologic detection. In plasma, TRX 80 may be present in protein dimers (Pekkari et al. (2000) (supra)). TRX also can be bound to other proteins such as albumin, hindering its detection in nondenaturing sample processing methods.
To clearly define a role for TRX in disease, and to use the detection of TRX and/or TRX 80 to diagnose a disease, monitor its progress, or the like, it is important to be able to simultaneously monitor the presence of both TRX forms in a quantitative capacity in complex samples, including plasma and tissue homogenates. There is a need for an assay that can detect the two forms of TRX, unambiguously and quantitatively, e.g. within the same sample.